mRNA荧光复合扩增系统鉴别正常和异常精液初探

目的构建mRNA荧光复合扩增体系,实现对不同种类精液(尤其是无精症精液)的区分鉴别。方法收集正常、少精症及无精症的精液样本,制备精斑样本后提取细胞总RNA,利用逆转录PCR技术扩增2个精子特异mRNA标记(PRM1、PRM2)、2个精浆特异mRNA标记(TGM4、SEMG1)和2个管家基因mRNA标记(TEF、UCE)。结果正常精液样本可检测到全部精液mRNA标记表达;少精症精液样本虽然可检测到全部mRNA标记表达,但精子特异mRNA标记表达量较低;无精症精液样本不能检测到精子mRNA标记,只能检测到精浆特异mRNA标记。结论利用mRNA荧光复合扩增系统可以实现对正常和无精症精液的区分,而正常精液和少精症精液相比差异无统计学意义。     

不同保存时间和不同精子数量精斑样本DNA分型比较

目的比较不同保存时间和不同精子数量精斑样本DNA分型的效果。方法制备精斑样本,保存10d的样本采用激光显微捕获30、20、15、10、5、1个精子,用于不同数量精子分型比较;保存10d、214d、375d的样本分别捕获30、20、10个精子,用于不同保存时间分型比较。比较各组检出率、等位基因丢失率和非特异性扩增率,采用χ2检验进行差异比较。结果①不同精子数量分型：捕获10个精子即可得到完整的DNA分型,且随着精子数增多,检出率逐渐提高而等位基因丢失率逐渐降低,30个精子等位基因丢失率为0%,1个精子则可达58.89%;②不同保存时间分型：总趋势是保存时间越短,捕获精子越多检出率越高,10个精子与20、30个精子组比较,均有显著性差异（P〈0.05）;等位基因丢失率及非特异性扩增率则随保存时间的延长而增加,相同保存时间的不同精子数量组之间和相同的精子数量的不同保存时间组之间比较,差异均具有统计学意义（P〈0.05）。结论激光显微捕获精子数目和检材保存时间对DNA分型结果有直接影响。     

人类精子ABO血型抗原的间接免疫荧光研究

应用间接免疫荧光技术,对40份精子标本进行 ABO 血型抗原检测。A 型人精子上存在 A 抗原;B 型人精子上存在 B 抗原;AB 型人精液中,一部分精子带有 A 抗原,另一部分精子带 B 抗原。各血型人的精子均有 H 抗原。精子血型抗原为本身所固有,并非来源于精浆。不同人的精子血型抗原含量各不相等,与其供体是否为分泌状态或强弱无直接关系。精子 ABO 血型抗原主要存在于精子的颈部和顶体等区域。     

人精浆中A型血型物质的分离纯化及理化性质研究

应用凝胶过滤、阴离子高速液相色谱及免疫亲合层析方法成功地从A型分泌型人精浆中分离、纯化了具有A型活性的精浆血型物质（SPBGS－A）。理化分析结果表明：SPBGS－A为表现单一A型活性、分子量不均一的糖蛋白。其分子量约为100KD；等电点在pH2．7～3．5之间；糖含量占72．5％；蛋白质含量占24．2％。共检出了16种氨基酸成分，其中苏氨酸、丝氨酸及脯氨酸的含量约占50％。经与其它水溶性血型物质在化学组成上进行比较，提示其可能具有一定的器官特异性。     

用胶乳凝集抑制试验鉴定人类精斑

作者用人类精浆致敏的胶乳和人初乳吸收后的抗人精液血清,采用胶乳凝集抑制试验凹玻板法盲测了180份生物性斑痕,结果表明此法确证人类精斑的敏感性,准确性均高于精子检出法,而与抗 p30血清琼脂双向扩散结果一致,且可用于混合斑的检验,具有快速、准确、简便等优点。     

上海某区非正常死亡统计学分析

按国际疾病标准分类法对1995-2004年间某区居民非正常死亡资料进行统计学分析。结果显示1995-2004年间年均非正常死亡率为4.6!(4.9!～4.5!),80岁以上组人群非正常死亡率最高;女性高于男性,为男性的1.03～1.36倍;意外跌落、交通事故、自杀为引起非正常死亡的前3位原因。表明非正常死亡已经成为严重影响某区居民健康、生命质量的主要因素,是目前较为重要的社会问题,应引起足够重视。     

大鼠死后肝脏磷酸果糖激酶-2含量的变化

目的 分析不同死亡时间和死亡原因大鼠肝脏组织内磷酸果糖激酶-2(PFK-2)含量的变化.方法 大鼠105只随机分为三组,以不同方式(失血、窒息、断颈)处死.分别于死后0、1、2、4、8、12和24h取大鼠肝脏组织,免疫组化和图像分析技术测定大鼠PFK-2变化.结果 三组不同死亡原因大鼠肝脏组织内PFK-2的含量随死亡时间延长呈规律性减弱趋势.结论 大鼠肝脏组织内PFK-2的变化可以反映死亡时间的变化趋势.     

免疫磁珠法与差异裂解法检验混合样本的效果观察

目的分析应用免疫磁珠分离技术和差异裂解法对混合样本的DNA检验效果,评价其在法医学应用中的价值。方法制备含有不同数量精细胞与阴道上皮细胞混合悬液和植绒混合斑拭子,分别采用MOSPD3抗体、SPAG8抗体免疫磁珠法及差异裂解法对样本进行检验,观察分析不同比例的混合细胞悬液,以及不同保存时间植绒混合斑拭子DNA分型的正确率。结果当混合细胞悬液内精细胞与阴道上皮细胞比例约1∶10时,免疫磁珠法的正确率明显高于差异裂解法(P0.01);当比例等于或高于1∶1时,两种方法正确率的差异无显著性意义(P0.05)。植绒拭子混合斑的免疫磁珠法正确率明显低于差异裂解法(P0.01);随混合斑保存时间的延长,差异裂解法正确率无明显下降(P0.05)。结论免疫磁珠法适用于新鲜混合样本中精细胞的分离检验,且在精细胞含量较低时优势更明显,差异裂解法较适合陈旧混合斑的分离检验。     

抗人精液血清特异性的研究

确证精斑常用抗人精液血清作沉淀反应。这种抗血清特异性差,常与人类某些体液发生交叉反应,以致精斑确证试验结果不可靠,影响鉴定结果的准确性。作者用人类精子、精浆、精液与人类精浆特异性抗原p30分别免疫家兔,制备各种抗血清,用环状沉淀、琼脂双向扩散及免疫电泳等技术对这些抗血清的特异性进行了研究,报道如下。     

新型滥用药物丁丙诺啡分析方法研究进展

本文介绍了丁丙诺啡的提取及检测现状,比较了液液提取、固相萃取、酶消化、衍生化等前处理方法的优劣和TLC、GC、GC/MS以及LC、LC/MS等不同分析方法的应用情况。     

Enantiomeric Identification of Pregabalin by GC‐MS via Methylation and S‐TPC Chiral Derivatization

Pregabalin is a Schedule V controlled substance which is defined as the (S) enantiomer of 3‐(aminomethyl)‐5‐methylhexanoic acid. It is used legitimately to treat neuropathy in patients with diabetes as well as for epilepsy and fibromyalgia. Pregabalin is an amino acid and an amphoteric compound, which makes it difficult to analyze using the conventional GC‐MS instrumentation found in most forensic drug analysis laboratories. Problems associated with the traditional GC‐MS analysis of pregabalin include selective solubility, ring closure to the corresponding lactam in the GC injection port and/or the MS transfer line and difficulty with chiral derivatization due to the presence of a carboxylic acid moiety. Here, we show that these challenges can be overcome by methylating (capping) the carboxylic acid portion of the pregabalin molecule and converting to the corresponding methyl ester. Once the methyl ester is synthesized, chiral derivatization at the amine can be achieved to identify the controlled (S) enantiomer of pregabalin via GC‐MS.     

GC/MS、GC/NPD法检测毛发中的氯胺酮

目的采用液-液萃取、衍生化和GC/MS、GC/NPD方法,进行毛发中氯胺酮定性定量分析。方法选择4-苯基丁胺为内标,毛发样本用NaOH、HCl及芳基硫酸酯酶/β-葡萄糖醛酸酶等3种方式进行水解,再进行衍生化后,采用GC/MS和GC/NPD方法定性定量分析。对不同水解和衍生化条件以及提取溶剂进行比较优化,并考察方法精密度、稳定性和检出限。结果方法的提取回收率大于95%,精密度和样品稳定性良好,日内和日间标准偏差小于6%;采用GC/NPD和GC/MS直接分析毛发中的氯胺酮,检出限为0.2ng/mg和2.0ng/mg,线性范围为10.0～250.0ng/mg,相关系数均大于0.99;采用酰化衍生化后分析,GC/NPD和GC/MS检出限分别提高至0.1ng/mg和0.2ng/mg。结论该方法回收率高、检测限低,可以用于毛发中氯胺酮的定性定量分析检验。     

Toward multidimensional information: A derivatization-free UHPLC-QqQ MS/MS method for amino acid components of fingerprint

The analysis of fingerprint chemical composition is a meaningful way to excavate the multidimensional information of fingerprint, including the donor profiling information and the age of a fingerprint, which broadens the evidential values of fingerprint, especially for the partial and distorted fingerprint. But the research remains still in the pilot phases or is ongoing. Amino acids are the dominant organic substances in latent sweat fingerprint and influenced by many donor factors. Hence, their content reflects personal information of donors. Forensic science will be revolutionized if suspects can be individualized by their amino acid content. The diverse nature, distinct physicochemical properties, and ultra-micro levels of amino acids present in fingerprints make it hard to detect. A high sensitivity method for detecting and quantifying multiple amino acid components is required. UHPLC-QqQ MS/MS offers high sensitivity, high separation, simultaneous multicomponents detection, and no derivatization, making it an ideal method for detecting and analyzing amino acids in fingerprints. Therefore, in this study, we propose and validate an efficient UHPLC-QqQ MS/MS method for the extraction and analysis of 13 amino acids from fingerprint. We compared the results of amino acids of 10 different substrates and found that the inherent amino acids in most porous substrates would have been extracted along with the fingerprint amino acids, making them unsuitable for quantitative amino acid analysis. Instead, plastic sheets are ideal substrates for laboratory studies. Then, extensive experiments were conducted among 30 donors for multidimensional information analysis. The type of samples analyzed were eccrine-rich fingerprints. A Binary Logistic Regression (BLR) model was developed, and the female and male donors were successfully differentiated by amino acids in fingerprints. Two other mathematical models were also developed to verify the accuracy, and all three different mathematical models were able to identify donors of different genders with over 90% accuracy. This demonstrates that amino acids have the potential to provide more information for donors as metabolic markers. In the future, we will conduct a series of experiments to analyze more multidimensional information for individual identification by amino acid content in the fingerprint.     

Free Fatty Acids Composition in Adipocere of the Kwäday Dän Ts’ìnchí Ancient Remains Found in a Glacier*

Abstract: Adipocere is a postmortem decomposition product consisting of mostly a mixture of free fatty acids (FFAs) that are formed because of the hydrolysis of triglycerides in adipose tissues. This article describes a simple and robust method for the extraction, identification, and quantification of FFA commonly found in adipocere using gas chromatography–mass spectrometry (GC/MS). This method was applied to analyze tissues from Kwäday Dän Ts’ìnchí, ancient remains discovered in a retreating glacier in the Tatshenshini‐Alsek Park, British Columbia, Canada in August 1999. The lyophilized tissues were grinded and extracted with hexane. The trimethylsilyl fatty acid derivatives were analyzed by GC/MS, and the relative abundances of myristic acid, palmitic acid, oleic acid, and stearic acid were determined. Milligram per gram levels of saturated fatty acids were found in the tissues of the ancient remains, while the levels of unsaturated fatty acids, such as palmitoleic acid, were found to be negligible. The results provided further evidence of the existence of adipocere found during forensic examination of the Kwäday Dän Ts’ìnchí ancient remains.     

GC／MS检测尿中4种苯氧羧酸类除草剂

目的建立尿中2，4-D、2，4-DP、MCPA、MCPP等4种苯氧羧酸类除草剂的分析方法；方法液液提取分离，乙醚为提取液，DCPA为内标，硫酸正丁醇酯化衍生化，气质联用分析法分析；结果尿中4种除草剂添加样品的相对回收率在80％以上，检测限都在5ng／ml以下，对中毒兔尿样进行了分析；结论对实际发生的中毒案件分析有足够的灵敏度。     

GC–MS analysis of eight aminoindanes using three derivatization reagents

Aminoindanes are a class of novel psychoactive substances (NPSs) that have become more prevalent over the past decade. GC–MS is often utilized for identifying seized drugs and is well regarded for its ability to separate mixtures. However, certain aminoindanes have similar mass spectral data and require specific gas chromatographic stationary phases for separation. Derivatization is an alternative method that can be applied to GC–MS to enhance chromatographic results, providing more selective analysis in seized-drug identification. This study investigates derivatization techniques to provide options for forensic science laboratories in accurately identifying aminoindanes. Three derivatization reagents, N-methyl-bis(trifluoroacetamide) (MBTFA), heptafluorobutyric anhydride (HFBA), and ethyl chloroformate (ECF) were evaluated for the analysis of eight aminoindanes by GC–MS using two common gas chromatographic stationary phases, Rxi®-5Sil MS and Rxi®-1Sil MS. All three derivatization methods successfully isolated eight aminoindanes, including the isomers 4,5-methylenedioxy-2-aminoindane (4,5-MDAI), and 5,6-methylenedioxy-2-aminoindane (5,6-MDAI) that could not be differentiated prior to derivatization. Reduced peak tailing and increased abundance were observed after derivatization for all the compounds, and mass spectra of the derivatives contained individualizing fragment ions that allowed for further characterization of the aminoindanes. This excluded 4,5-MDAI and 5,6-MDAI as they shared the same characteristic ions and were only distinguishable by their retention times. All three derivatization techniques used in this study allow for successful characterization of the aminoindanes and give forensic science laboratories flexibility in their analysis approach when they encounter these compounds.     

Analysis of drugs of abuse in hair by automated solid-phase extraction, GC/EI/MS and GC ion trap/CI/MS

In our laboratory, analysis of human hair for the detection of drugs of abuse was first performed in 1995. Initially, requests for hair analysis were few, and it is only since 1997 that these analyses have become routine. As demand grew, we developed an automatic solid-phase extraction method; the use of a robot ASPEC allowed us to drop certain fastidious manipulations, and to treat a large number of samples at a time. This method is described, along with analysis by gas-chromatography-mass spectrometry (GC/MS) in selected ion monitoring mode (SIM), for the following drugs: codeine, 6-monoacetylmorphine (6-MAM), morphine, cocaine, methadone, ecstasy (MDMA) and Eve (MDE). This requires prior derivatization with propionic anhydride. The different validation parameters, linearity, repeatability, recovery and detection limits are described, as well as the application of this method to some real cases. Analysis of these cases is also performed by an ion trap GC/MS in chemical ionization mode (GC/IT/CI/MS) in order to demonstrate the usefulness of this technique as a complement to routine analysis. Analysis by GC/IT/CI/MS indeed avoids the risk of false-positive results by the identification of metabolites.     

The detection of psilocin in human urine

Pharmacokinetic studies of psilocybin in humans have shown the rapid dephosphorylation of psilocybin to psilocin with further conversion to 4-hydroxy-tryptophole (4HT) and 4-hydroxyindole-3-acetic acid (4HIAA) in plasma. Our study shows that psilocin also undergoes conjugation and can be found in the urine as the psilocin-glucuronide conjugate. Recoveries after enzymatic hydrolysis of the urine with beta-glucuronidase (Helix Pomatia or E. Coli) when compared to non-hydrolyzed urine confirmed the presence of the glucuronide. Detection of psilocin from hydrolyzed and extracted samples was optimized for GC/MS by derivatization with MSTFA. The method developed allows for the detection of psilocin in urine with a limit of quantitation of 10 ng/mL, based on 5 mL of spiked urine. Using this method, our laboratory has confirmed the presence of psilocin in 6 out of 8 urine samples, with concentrations ranging from 10 ng/mL to greater than 200 ng/mL. Before implementation of the hydrolysis and derivatization steps, our limit of detection was 200 ng/mL, based on spiked urine standards. No case samples were positive without hydrolysis and derivatization.     

生物试样中巴比妥类药物固相提取柱上衍生化GC/MS分析

建立生物试样中常见巴比妥类药物的固相提取和柱上衍生化GC／MS分析法。将预制的血或肝分别在pH6．0和pH2.2的条件下过预活化的GDX－403吸附小柱，再用缓冲浪和蒸馏水各4ml顺序洗柱。最后用4ml丙酮／氯仿（1：1）溶剂洗脱样品，离心弃除水相，80℃挥至近干，用50μl乙醇定溶、取净化的样品2～4μl挥至近干，加20μl0．2molTMAH衍生化试剂，直接进样0.5μl，柱上衍生化GC／MS（GC）分析。在试验条件下，当血和肝分别添加2．0μg和5．0μg混合药物，回收率≥80％，相对标准差（RSD）优于±10％，检测限优于5ng（信／噪比≥2）。该法能有效地排除类脂物和组胺的干扰，可用于治疗量级药物分析和婴幼儿中毒案检验。